Antibodies to macrophage stimulating protein, bioassay and immunopurification method

ABSTRACT

A method of purifying macrophage stimulating protein. Antibodies to macrophage stimulating protein and a bioassay for the detection of antibodies which bind macrophage stimulating protein.

BACKGROUND OF THE INVENTION

This application is a divisional of application Ser. No. 07/586,085,filed Sep. 21, 1990, now U.S. Pat. No. 5,219,991.

FIELD OF THE INVENTION

The present invention relates to macrophage stimulating protein (MSP).In particular, the present invention relates to the purification of MSP,to highly purified MSP and antibodies specific thereto.

BACKGROUND INFORMATION

Macrophage stimulating protein (MSP), a component of mammalian bloodplasma, makes mouse peritoneal macrophages responsive tochemoattractants such as complement C5a [Leonard et al., Exp. Cell Res.102:434 (1976) and Leonard et al., Exp. Cell Res. 114:117 (1978)]. MSPalso stimulates mouse macrophages to increase their movement andpinocytic activity.

The discovery of MSP was based on the observation that peritonealmacrophages in RPMI 1640 medium did not migrate to C5a unless serum wasadded to the cell suspension. Sera from different mammals, includingmice and man, caused activation.

The low concentration of MSP in serum (less than 1/10⁵ of total serumprotein) precluded complete purification by conventional techniques.However, highly purified MSP is required for use in human treatments. Amethod resulting in highly purified MSP is desirable.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide amethod for homogeneous purification of MSP.

It is another object of the present invention to provide purified MSPand antibodies specific thereto. The highly purified MSP and/orantibodies thereto may be used for detection of MSP deficiency states,treating pathogen infections and treatment of disease characterized bymacrophage-mediated inflammation.

Various other objects and advantages of the present invention will beapparent to one of ordinary skill in the art from the drawings and thefollowing description of the invention.

In one embodiment, the present invention relates to homogeneousmacrophage stimulating protein characterized by a band of 70 kilodaltonson non-reducing sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), bands of 47 kilodaltons and 22 kilodaltonson reducing SDS-PAGE, the amino acid composition according to Table IIand a specific activity of 2×10⁵ units MSP/mg protein.

In another embodiment, the present invention relates to a monoclonalantibody to MSP of the present invention.

In a further embodiment, the present invention relates to a method ofpurifying macrophage stimulating protein comprising the application ofhuman serum with a specific activity of about 6 units MSP per mg proteinto an anti-MSP immunoaffinity column. After MSP is eluted from thecolumn, it is purified to a specific activity of 2×10⁵ units/mg protein.

In another embodiment, the present invention relates to a pharmaceuticalcomposition for the treatment of pathogen infection comprising thehighly purified MSP of the present invention in a pharmaceuticallyacceptable carrier, in an amount sufficient to stimulate macrophageactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B shows CM-HPLC purification of MSP. FIG. 1A: chromatographyof MSP eluted from immunoaffinity column. FIG. 1B: re-run of MSP peak.

FIG. 2 shows SDS-PAGE of purified HPLC-CM-2 MSP under non reducing(lanes A, B and C) and reducing (lanes D, E and F) conditions. Theamounts applied per lane were 125, 250 and 500 ng respectively for lanesA, B, C and D, E, F.

FIG. 3 shows the partial amino acid sequences of MSP fragments.

FIGS. 4A-4D shows response of mouse resident macrophages in tissueculture dishes to a 1 hr incubation with pure MSP. FIGS. 4A through 4D:0, 10⁻¹¹ M, 10⁻¹⁰ M, and 10⁻⁹ M, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in part to a method of isolating MSP in ahighly pure state. Starting with human serum (having a specific activityof about at least 6 units MSP/mg protein), this invention provides ameans of obtaining highly purified MSP. The method involves purificationof human serum MSP on an immunoaffinity column of anti-MSP, followed bycarboxy methyl high performance liquid chromatography (CM-HPLC), in twosequential runs. The increase in purity resulting from each step of themethod of the present invention is shown in Table I as increase inspecific activity. The eluted pure MSP is characterized by a specificactivity of about 2×10⁵ units MSP/mg protein. Utilizing this method, MSPhas been purified to homogeneity as determined by SDS-PAGE and partialamino acid sequencing of reduced and alkylated α and β chains.

The highly purified MSP is characterized by a band of 70 kilodaltons onnon-reducing SDS-PAGE and bands of 47 kilodaltons and 22 kilodaltons onreducing SDS-PAGE. In addition, the MSP has the amino acid compositiongiven in Table II. Further, the highly purified MSP has a specificactivity of about 2×10⁵ units MSP/mg protein.

Peptide fragments of the purified MSP have been sequenced and are shownin FIG. 3. The sequences were compared with the sequences in the ProteinData Bank. The amino acid composition distinguishes the MSP from otherknown proteins.

The present invention also relates to antibodies (both monoclonal andpolyclonal) to MSP. Examples of antibodies belonging to the presentinvention are the mouse monoclonal IgG anti-MSP antibodies used in theimmunoaffinity column (see Examples). Accordingly, the present inventionfurther relates to antibodies to MSP bound to a solid support such asSepharose gels or polystyrene.

The present invention further relates to bioassays for the detection ofMSP or antibodies specific therefor present in a sample such as, forexample, a serum sample. Many types of tests, as one skilled in the artwill appreciate, can be used for detection. Such tests include, but arenot limited to, ELISA.

In an embodiment of the bioassay of the present invention, a surface(ie. a solid support) is coated with the highly purified MSP of thepresent invention and contacting with the sample (eg. serum). Thepresence of a resulting complex formed between the coated surface andantibodies specific therefor in the sample can be detected by any of theknown methods common in the art, such as an enzyme-conjugated anti-IgG.

The following non-limiting examples are provided to aid in theunderstanding of the present invention, the true scope of which is setforth in the appended claims. It is understood that modifications can bemade in the procedure set forth, without departing from the true spiritof the invention.

EXAMPLES

Throughout the following procedure, MSP was monitored by bioassay [Falket al., J. Immunol. Methods 37:39 (1980) and Leonard et al., J.Reticuloendothelial Soc. 30:271 (1981)]. Screening for hybridomaantibody was by inhibition of biological activity [Leonard et al., J.Immunol. Methods 82:341 (1985)].

Purification of MSP

Step 1

MSP bioassay

The assay for MSP is based on a concentration-dependent increase in thein vitro chemotactic response of resident peritoneal C3H mousemacrophages to endotoxin-activated mouse serum [Leonard et al., Exp.Cell Res. 102:434 (1976)]. Resident macrophages from peritoneal cavitiesof normal C3H mice were obtained by lavage with 7-8 ml of RPMI 1640medium containing 2% BSA. Cells were centrifuged at 4° C. in 50 mlpolypropylene tubes for 10 min at 250×g, and resuspended in RPMI 1640without added protein at a macrophage concentration of 10⁶ /ml. For thebioassay, bottom wells of a multiwell chemotaxis chamber [Falk et al.,J. Immunol. Methods 37:39 (1980)] were filled with chemoattractant (a1/200 dilution of endotoxin activated mouse serum) and covered with a 10μm thick polycarbonate membrane with 5 μm holes. After gasket and topplate were added to complete assembly of the chamber, the upper wellswere filled with 50 μl volumes of macrophage suspensions in RPMI 1640medium containing the stimulating protein to be assayed. During theincubation period of 3 hrs at 37° C in humidified air with 5% CO₂,macrophages migrated through holes in the membrane and remained attachedto the attractant side of the membrane. The chamber was thendisassembled, and cells were wiped away from the non-migrated side ofthe membrane. After air drying and staining, migrated cells were countedwith an image analyzer [Leonard et al., J. Reticuloendothelial Soc.30:271 (1981)]. A unit of MSP per ml of test solution was defined as thereciprocal of the dilution required to induce approximately 30% of themaximal chemotactic response obtained at the plateau of thedose-response curve.

Isolation of MSP Antigen for Monoclonal Antibody Production

Large-scale purification of MSP from 3600 ml of outdated frozen plasma(Blood Bank, Clinical Center, NIH, Bethesda) was based in part onpublished methods [Leonard et al., Exp. Cell Res. 114:117 (1987)].Plasma was lyophilized and reconstituted to 1/3 of the starting volume.Eight grams of Na₂ SO₄ per 100 ml of concentrated plasma at 20° C. wereadded to precipitate unwanted high molecular weight proteins.Supernatant was separated from precipitated protein by centrifugation at4° C. and divided into 12 aliquots for gel filtration on Sephadex G-200.Fractions with MSP activity eluted from Sephadex G-200 in a regioncorresponding to a molecular mass of about 100 kD. Pooled fractions fromthe 12 column runs (1800 ml total) were dialyzed against pH 8.0, 0.005Mpotassium phosphate buffer and applied to a column containing 170 gmDEAE-cellulose. Unbound protein in the pass-through volume wasdiscarded, and MSP was eluted by a step increase in NaCl concentrationto 0.045M in starting buffer. The 380 ml MSP pool was prepared forelectrofocusing by lyophilization and reconstitution with water to avolume of 57 ml. This was divided into 3 aliquots for 3 runs on anelectrofocusing flat bed. Electrofocused fractions with MSP activitywere in the pH range 7.0-7.5. Pooled MSP from each run was dialyzedagainst 0.0025 M potassium phosphate, pH 7.4, lyophilized, reconstitutedwith 20 mg/ml sucrose, and run on a 16-550 mm column of Sephadex G-200.Pooled MSP from the 3 Sephadex runs was in a volume of 56 ml, with anA₂₈₀ of 1.5. It was lyophilized, reconstituted with 3 ml of water, andequilibrated with formalin-fixed Cowan strain S. aureus (ZymedLaboratories, San Francisco, Calif.) to absorb out traces of IgG.

Step 2

Immunization of Mice, Production of Monoclonal Anti-MSP, and Preparationof Immunoaffinity Column

MSP, partially purified as described above and emulsified in completeFreund's adjuvant, was injected intraperitoneally into BALB/c mice. Theamount of protein per injection was 280 μg. MSP in incomplete Freund'sadjuvant was injected subcutaneously 2 weeks later. The following week,serum from the mice was tested for anti-MSP activity by the capacity ofcolumn-bound serum to absorb applied MSP [Leonard et al., J. Immunol.Methods 82:341 (1985)]. Two weeks after this, 0.1 ml of MSP solution wasinjected intravenously; spleens of the two mice with the most anti-MSPactivity were removed four days later. Fusion, cloning and propagationof cell hybrids were done by published methods [Schowalter et al., Inf.Immun. 34:684 (1981)]. Beginning at about 10 days, supernatants from thehybrid cells were tested by an immunoabsorbance assay [Leonard et al.,J. Immunol. Methods 82:341 (1985)] for anti-MSP activity. Cells frompositive wells were cloned. Several individual clones were obtained thatproduced IgG anti-MSP. After passage of these clones intraperitoneallyin mice, IgG was purified from ascitic fluid on Protein-A Sepharose. Theimmunoaffinity column for the first purification step was made by theaddition of 240 mg of IgG monoclonal anti-MSP to protein-A Sepharose.The column had a diameter of 2.5 cm and a bed height of 8 cm.

Step 3

Purification of MSP Immunoaffinity and HPLC Chromatography

Four 6 liter batches of frozen, outdated human plasma were used forlarge scale purification of MSP. A 6 liter batch was thawed, filteredthrough layers of sterile gauze squares, centrifuged at 20° C. for 15min at 10,000 rpm in a JA-10 rotor in a Beckman J2-21 centrifuge,filtered again through gauze to remove floating lipid, and finallyfiltered through a glass fiber prefilter (Nalgene 280-5000, Nalge Co.,Rochester, N.Y.) supported on a stainless steel mesh on a Milliporesuction funnel. The volume of clean plasma was about 5 liters. Plasmawas run through the column with a hydrostatic head of about 3 meters,over a period of 4 hrs at 20° C. After the column was rinsed with 0.15MNaCl with 0.05M tris buffer, pH 8.0, MSP was eluted with 0.1M, pH 2.5glycine buffer. Fractions of 5 ml were collected into tubes containing 1ml of 0.5M, pH 7.9 potassium phosphate buffer. The A₂₈₀ peak, comprisingabout 110 ml, was dialyzed at 4° C. overnight against 0.03M NaCl, 0.02M3-[N-morpholino]propanesulfonic acid, pH 6.7, which was the startingbuffer for CM-HPLC. It was centrifuged at 20,000 rpm in a Type 30 rotorof a Beckman L8-70 ultracentrifuge at 5° C., concentrated on an AmiconYM-10 ultrafilter to 2 ml, dialyzed again against starting buffer, andfiltered through a 5 ml, 0.45 μm Centrex unit (Schleicher and Schuell,Keene, N.H.). It was then applied to a Beckman Spherogel-TSK CM-3SW dp10 μm column (7.5 mm×7.5 cm). Running conditions were flow of 1 ml/min,2 min/fraction, A₂₈₀ absorbance range of 1.28. After the flow-through, alinear gradient of NaCl in starting buffer was run to a limitconcentration of 0.3M. Fractions were assayed for MSP biologicalactivity. About 10 fractions were pooled and froze across the MSP peak(HPLC-CM-1 MSP), which was in the region of 0.2M NaCl (FIG. 1, toppanel). After 3 more 5 liter batches of plasma were processed as above,the 4 HPLC-CM-1 MSP peaks were pooled, dialyzed against HPLC-CM startingbuffer, concentrated to 2 ml, dialyzed against starting buffer again,filtered, and chromatographed on the HPLC-CM column under the sameconditions as the first run. Fractions across the A₂₈₀ peak were pooled(HPLC-CM-2 MSP, FIG. 1, bottom panel), to yield pure MSP. Table 1 shownbelow is a summary of purification and yield of MSP.

                  TABLE I                                                         ______________________________________                                        Purification and yield of MSP                                                                     units MSP/                                                            mg protein                                                                            mg protein total MSP                                      ______________________________________                                        5 liters human plasma                                                                       35 × 10.sup.4                                                                     6          2 × 10.sup.6                         Anti-MSP column eluate                                                                      67        3 × 10.sup.3                                                                       2 × 10.sup.5                         HPLC-CM-1 eluate                                                                            1.5       7 × 10.sup.4                                                                       1 × 10.sup.5                         HPLC-CM-2 eluate                                                                            0.6       2 × 10.sup.5                                                                       1 × 10.sup.5                         ______________________________________                                    

This Table shows that in the first immunoaffinity column step specificactivity of MSP increased from 6 to 3000, and total protein decreasedfrom 350 g to about 70 mg. However, MSP was still only about 1 percentof the total protein in this product, as illustrated by drop from 67 to0.6 mg (with a minimal decrease in total yield) in the HPLC-CM steps.

Characterization of Purified MSP

MSP was characterized by SDS-PAGE under reducing and non-reducingconditions; Western blot analysis, with monoclonal anti-MSP; reductionand alkylation of the α and β chains of MSP, followed by chainseparation by gel filtration; amino acid analysis of native MSP, as wellas α and β chains; partial amino acid sequence analysis of cyanogenbromide treated MSP, of alkylated MSP β chain, and of purifiedlysylendopeptidase digest fragments of MSP α and β chains.

Purified MSP was run on SDS-PAGE under non-reducing (lanes A,B,C,) andreducing (lanes D,E,F) conditions (FIG. 2). The amounts applied per lanewere 125, 250, and 500 ng. It was concluded that MSP comprises 2disulfide-linked chains with molecular masses of approximately 47 and 22kd. Monoclonal anti-MSP reacted with protein corresponding to the bandfound on SDS-PAGE under non-reducing conditions. Reactivity wasundetectable or minimal with MSP on SDS-PAGE under reducing conditions.

Amino acid composition of MSP and its two chains is shown below in TableII. Partial sequence data are shown in FIG. 3.

The 6 sequences with the fewest ambiguities (indicated by asterisks inFIG. 3) were compared with sequences in the Protein Data Bank. Two ofthe 6 fragments had highly significant sequence similarities to twoknown proteins. The first 12 residues of α chain fragment BU-12 matchedperfectly with residues 162-173 of bovine prothrombin. BU-12 had only2/12 mismatches with residues 154-165 and with the approximatelyrepeating residues 259-270 of human prothrombin. The 10-residue β chainfragment BU-5 corresponded, with 3 mismatches, to residues 689-698 ofhuman plasminogen. The partial sequences of MSP comprise about 15percent of the total molecule. The fragments noted have sequencesimilarity to two zymogens associated with coagulation systems.

As shown in Table I, pure MSP makes mouse resident peritonealmacrophages responsive to chemoattractant, with an EC₃₀ of about 6×10⁻¹¹M. FIG. 4 shows the response of mouse resident peritoneal macrophages toa 1 hr incubation with pure MSP. Frames a through d: 0, 10⁻¹¹ M, 10⁻¹⁰ Mand 10⁻⁹ M MSP. Greatly elongated cell processes and increased numbersof pinocytic vesicles are seen.

                  TABLE II                                                        ______________________________________                                        Amino Acid Composition of MSP.                                                        Residues/mole.sup.1                                                   Amino Acid                                                                              Native MSP  α Chain                                                                              β Chain                               ______________________________________                                        D         45          35           12                                         E         65          39           24                                         S         33          21           16                                         G         117         78           54                                         H         17          12           4                                          R         33          26           9                                          T         41          26           10                                         A         59          40           17                                         P         64          40           14                                         Y         11          11           4                                          V         52          25           16                                         M         8           4            3                                          I         13          7            6                                          L         47          26           15                                         F         18          14           5                                          K         22          16           7                                          Total     645         420          216                                        ______________________________________                                         .sup.1 Based on estimated molecular masses from SDS PAGE of 70, 48 and 22     kDa for native MSP, α chain and β chain.                      

Statement of Deposit

A hybridoma producing mouse monoclonal IgG anti-MSP antibody wasdeposited on Aug. 6, 1990 at the American Type Culture Collection 12301Parklawn Drive, Rockville, Md. 20852. The hybridoma has been assignedthe ATCC accession number HB 10522. The immunoaffinity column for thepurification of MSP can be made with the antibody produced by thishybridoma.

All publications mentioned hereinabove are hereby incorporated byreference.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention.

What is claimed is:
 1. A purified antibody which binds MacrophageStimulating Protein wherein said macrophage stimulating protein ischaracterized as having a molecular weight of 70 kilodaltons asdetermined by non-reducing sodium dodecyl sulfate polyacrylamide gelelectrophoresis, and the subunits have molecular weights of 47 kD and 22kD as determined by reducing sodium dodecyl sulphate polyacrylamide gelelectrophoresis, and a specific activity of at least 2×10⁵ unitsmacrophage stimulating protein/mg protein.
 2. An antibody according toclaim 1 which is monoclonal.
 3. An antibody according to claim 1 whichis polyclonal.
 4. A complex comprising said antibody according to claim1 bound to a solid support.
 5. A method of purifying macrophagestimulating protein comprising treating partially purified macrophagestimulating protein having a specific activity of at least 6 unitsmacrophage stimulating protein/mg protein by anti-macrophage stimulatingprotein immunoaffinity column purification followed by high performanceliquid chromatography and eluting therefrom macrophage stimulatingprotein which is characterized by a specific activity of at least 2×10⁵units macrophage stimulating protein/mg protein.
 6. A monoclonalantibody according to claim 2 wherein the antibody binds purifiedmacrophage stimulating protein on sodium dodecyl sulphate polyacrylamidegel electrophoresis under non-reducing conditions.
 7. The monoclonalantibody produced by the hybridoma ATCC HB10522.
 8. A bioassay for thedetection of antibodies for macrophage stimulating protein comprisingthe steps of:(a) coating a surface with purified macrophage stimulatingprotein wherein said macrophage stimulating protein is characterized ashaving a molecular weight of 70 kilodaltons as determined bynon-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis,and the subunits have molecular weights of 47 kilodaltons and 22kilodaltons as determined by reducing sodium dodecyl sulphatepolyacrylamide gel, and a specific activity of at least 2×10⁵ unitsmacrophage stimulating protein/mg protein; (b) contacting said coatedsurface with an antibody containing sample; and (c) detecting a complexformed between said protein and antibodies in said sample.